Peptide microarray

The assay principle of peptide microarrays is similar to an ELISA protocol.

The peptides (up to tens of thousands in several copies) are linked to the surface of a glass chip typically the size and shape of a microscope slide. This peptide chip can directly be incubated with a variety of different biological samples like purified enzymes or antibodies, patient or animal sera, cell lysates and then be detected through a label-dependent fashion, for example, by a primary antibody that targets the bound protein or modified substrates. After several washing steps a secondary antibody with the needed specificity (e.g. anti IgG human/mouse or anti phosphotyrosine or anti myc) is applied. Usually, the secondary antibody is tagged by a fluorescence label that can be detected by a fluorescence scanner.{{cite journal |pmid=15384422 |year=2004 |last1=Panse |first1=S |last2=Dong |first2=L |last3=Burian |first3=A |last4=Carus |first4=R |last5=Schutkowski |first5=M |last6=Reimer |first6=U |last7=Schneider-Mergener |first7=J |title=Profiling of generic anti-phosphopeptide antibodies and kinases with peptide microarrays using radioactive and fluorescence-based assays |volume=8 |issue=3 |pages=291–9 |journal=Molecular Diversity |doi=10.1023/B:MODI.0000036240.39384.eb|s2cid=407457 }} Other label-dependent detection methods includes chemiluminescence, colorimetric or autoradiography.

Label-dependent assays are rapid and convenient to perform, but risk giving rise to false positive and negative results.{{Cite journal|last1=Kaeberlein|first1=Matt|last2=McDonagh|first2=Thomas|last3=Heltweg|first3=Birgit|last4=Hixon|first4=Jeffrey|last5=Westman|first5=Eric A.|last6=Caldwell|first6=Seth D.|last7=Napper|first7=Andrew|last8=Curtis|first8=Rory|last9=DiStefano|first9=Peter S.|date=2005-04-29|title=Substrate-specific Activation of Sirtuins by Resveratrol|journal=Journal of Biological Chemistry|volume=280|issue=17|pages=17038–17045|doi=10.1074/jbc.M500655200|issn=0021-9258|pmid=15684413|doi-access=free}} More recently, label-free detection including surface plasmon resonance (SPR) spectroscopy, mass spectrometry (MS) and many other optical biosensors{{Cite journal|last1=Fernández Gavela|first1=Adrián|last2=Grajales García|first2=Daniel|last3=Ramirez|first3=Jhonattan C.|last4=Lechuga|first4=Laura M.|authorlink4=Laura Lechuga|date=2016-02-24|title=Last Advances in Silicon-Based Optical Biosensors|journal=Sensors|volume=16|issue=3|pages=285|doi=10.3390/s16030285|pmid=26927105|pmc=4813860|bibcode=2016Senso..16..285F |doi-access=free}}{{Cite book|title=Optical Guided-wave Chemical and Biosensors II|volume = 8|last=Fang|first=Ye|date=2010|publisher=Springer, Berlin, Heidelberg|isbn=9783642028267|series=Springer Series on Chemical Sensors and Biosensors|pages=27–42|doi=10.1007/978-3-642-02827-4_2|chapter = Resonant Waveguide Grating Biosensor for Microarrays}}{{Cite journal|last1=Picaud|first1=Sarah|last2=Filippakopoulos|first2=Panagis|date=2015-08-17|title=SPOTing Acetyl-Lysine Dependent Interactions|journal=Microarrays|volume=4|issue=3|pages=370–388|doi=10.3390/microarrays4030370|pmid=27600229|pmc=4996381|doi-access=free}}{{Cite journal|last1=Hundsberger|first1=Harald|last2=Önder|first2=Kamil|last3=Schuller-Götzburg|first3=Peter|last4=Virok|first4=Dezso P.|last5=Herzog|first5=Julia|last6=Rid|first6=Raphaela|date=2017-06-08|title=Assembly and use of high-density recombinant peptide chips for large-scale ligand screening is a practical alternative to synthetic peptide libraries|journal=BMC Genomics|volume=18|issue=1|pages=450|doi=10.1186/s12864-017-3814-3|pmid=28595602|pmc=5463365|issn=1471-2164 |doi-access=free }} have been employed to measuring a broad range of enzyme activities.{{Cite journal|last1=Szymczak|first1=Lindsey C.|last2=Kuo|first2=Hsin-Yu|last3=Mrksich|first3=Milan|date=2018-01-02|title=Peptide Arrays: Development and Application|journal=Analytical Chemistry|volume=90|issue=1|pages=266–282|doi=10.1021/acs.analchem.7b04380|pmid=29135227|pmc=6526727|issn=0003-2700}}

Peptide microarrays show several advantages over protein microarrays:

• Ease and cost of synthesis
• Extended shelf stability
• Detection of binding events on epitope level, enabling study of i.e. epitope spreading
• Flexible design for peptide sequence (i.e. posttranslational modifications, sequence diversity, non-natural amino acids ...) and immobilization chemistries
• Higher batch-to-batch reproducibility

A peptide microarray is a planar slide with peptides spotted onto it or assembled directly on the surface by in-situ synthesis. Whereas peptides spotted can undergo quality controls that include mass spectrometer analysis and concentration normalization before spotting and result from a single synthetic batch, peptides synthesized directly on the surface may suffer from batch-to-batch variation and limited quality control options. However, peptide synthesis on chip allows the parallel synthesis of tens of thousands of peptides providing larger peptide libraries paired with lower synthesis costs.{{cite journal|last1=Beyer|first1=M|last2=Nesterov|first2=A|last3=Block|first3=I|last4=König|first4=K|last5=Felgenhauer|first5=T|last6=Fernandez|first6=S|last7=Leibe|first7=K|last8=Torralba|first8=G|last9=Hausmann|first9=M|last10=Trunk|first10=U|last11=Lindenstruth|first11=V|last12=Bischoff|first12=FR|last13=Stadler|first13=V|last14=Breitling|first14=F|title=Combinatorial synthesis of peptide arrays onto a microchip.|journal=Science|date=Dec 21, 2007|volume=318|issue=5858|pages=1888|pmid=18096799|doi=10.1126/science.1149751|bibcode=2007Sci...318.1888B|s2cid=33941406}} Peptides are ideally covalently linked through a chemoselective bond leading to peptides with the same orientation for interaction profiling. Some alternative procedures describe unspecific covalent binding and adhesive immobilization.

However, lithographic methods can be used to overcome the problem of excessive number of coupling cycles. Combinatorial synthesis of peptide arrays onto a microchip by laser printing has been described,{{Cite journal|last1=Breitling|first1=Frank|last2=Felgenhauer|first2=Thomas|last3=Nesterov|first3=Alexander|last4=Lindenstruth|first4=Volker|last5=Stadler|first5=Volker|last6=Bischoff|first6=F. Ralf|date=2009-03-23|title=Particle-Based Synthesis of Peptide Arrays|journal=ChemBioChem|volume=10|issue=5|pages=803–808|doi=10.1002/cbic.200800735|pmid=19191248|s2cid=13173295|issn=1439-7633}} where a modified colour laser printer is used in combination with conventional solid-phase peptide synthesis chemistry.{{Cite journal|last1=Stadler|first1=Volker|last2=Felgenhauer|first2=Thomas|last3=Beyer|first3=Mario|last4=Fernandez|first4=Simon|last5=Leibe|first5=Klaus|last6=Güttler|first6=Stefan|last7=Gröning|first7=Martin|last8=König|first8=Kai|last9=Torralba|first9=Gloria|date=2008-09-01|title=Combinatorial Synthesis of Peptide Arrays with a Laser Printer|journal=Angewandte Chemie International Edition|volume=47|issue=37|pages=7132–7135|doi=10.1002/anie.200801616|pmid=18671222|issn=1521-3773}} Amino acids are immobilized within toner particles, and the peptides are printed onto the chip surface in consecutive, combinatorial layers. Melting of the toner upon the start of the coupling reaction ensures that delivery of the amino acids and the coupling reaction can be performed independently. Another advantage of this method is that each amino acid can be produced and purified separately, followed by embedding it into the toner particles, which allows long-term storage.

Peptide microarrays can be used to study different kinds of protein-protein interactions, specially those involving modular protein substructures called peptide recognition modules or, most commonly, protein interaction domains. The reason for this is that such protein substructures recognize short linear motifs often exposed in natively unstructured regions of the binding partner, such that the interaction can be modelled in vitro by peptides as probes and the peptide recognition module as analyte. Most publications can be found in the context of immune monitoring and enzyme profiling.

• Mapping of immunodominant regions in antigens or whole proteomes{{cite journal|last1=Zandian|first1=Arash|last2=Forsström|first2=Björn|last3=Häggmark-Månberg|first3=Anna|last4=Schwenk|first4=Jochen M.|last5=Uhlén|first5=Mathias|last6=Nilsson|first6=Peter|last7=Ayoglu|first7=Burcu|title=Whole-Proteome Peptide Microarrays for Profiling Autoantibody Repertoires within Multiple Sclerosis and Narcolepsy|journal=Journal of Proteome Research|date=9 February 2017|volume=16|issue=3|pages=1300–1314|doi=10.1021/acs.jproteome.6b00916|pmid=28121444|doi-access=free}}{{cite journal |doi=10.1016/j.jaci.2009.05.024 |title=Development of a novel peptide microarray for large-scale epitope mapping of food allergens |year=2009 |last1=Lin |first1=Jing |last2=Bardina |first2=Ludmilla |last3=Shreffler |first3=Wayne G. |last4=Andreae |first4=Doerthe A. |last5=Ge |first5=Yongchao |last6=Wang |first6=Julie |last7=Bruni |first7=Francesca M. |last8=Fu |first8=Zhiyan |last9=Han |first9=Youngshin |last10=Sampson |first10=Hugh A. |journal=Journal of Allergy and Clinical Immunology |volume=124 |issue=2 |pages=315–22, 322.e1–3 |pmid=19577281 |pmc=2757036|display-authors=8 }}{{cite journal |pmid=22349330 |year=2012 |last1=Linnebacher |first1=M |last2=Lorenz |first2=P |last3=Koy |first3=C |last4=Jahnke |first4=A |last5=Born |first5=N |last6=Steinbeck |first6=F |last7=Wollbold |first7=J |last8=Latzkow |first8=T |last9=Thiesen |first9=HJ |last10=Glocker |first10=Michael O. |title=Clonality characterization of natural epitope-specific antibodies against the tumor-related antigen topoisomerase IIa by peptide chip and proteome analysis: A pilot study with colorectal carcinoma patient samples |volume=403 |issue=1 |pages=227–38 |doi=10.1007/s00216-012-5781-5 |journal=Analytical and Bioanalytical Chemistry|s2cid=33847079 |display-authors=8 }}{{Cite journal|last1=Jaenisch|first1=Thomas|last2=Heiss|first2=Kirsten|last3=Fischer|first3=Nico|last4=Geiger|first4=Carolin|last5=Bischoff|first5=F. Ralf|last6=Moldenhauer|first6=Gerhard|last7=Rychlewski|first7=Leszek|last8=Sié|first8=Ali|last9=Coulibaly|first9=Boubacar|date=April 2019|title=High-density Peptide Arrays Help to Identify Linear Immunogenic B-cell Epitopes in Individuals Naturally Exposed to Malaria Infection|journal=Molecular & Cellular Proteomics|volume=18|issue=4|pages=642–656|doi=10.1074/mcp.RA118.000992|doi-access=free |issn=1535-9484|pmc=6442360|pmid=30630936}}
• Seromarker discovery{{cite journal |doi=10.1038/news.2011.541 |title=Clues emerge to explain first successful HIV vaccine trial |year=2011 |last1=Callaway |first1=Ewen |journal=Nature}}{{cite journal |doi=10.1021/acs.jproteome.0c00484 |title=Rapid Response to Pandemic Threats: Immunogenic Epitope Detection of Pandemic Pathogens for Diagnostics and Vaccine Development Using Peptide Microarrays |year=2020 |last1=Heiss|first1=Kirsten|last2=Heidepriem|first2=Jasmin|last3=Fischer|first3=Nico|last4=Weber|first4=Laura K.|last5=Dahlke|first5=Christine|last6=Jaenisch|first6=Thomas|last7=Loeffler|first7=Felix F.|date=September 2020| journal=Journal of Proteome Research|volume=19 |issue=11 |pages=4339–4354 |pmid=32892628 |hdl=21.11116/0000-0007-05EF-7|hdl-access=free}}
• Monitoring of clinical trials{{cite journal |pmid=18481290 |year=2008 |last1=Garren |first1=H |last2=Robinson |first2=WH |last3=Krasulová |first3=E |last4=Havrdová |first4=E |last5=Nadj |first5=C |last6=Selmaj |first6=K |last7=Losy |first7=J |last8=Nadj |first8=I |last9=Radue |first9=EW |last10=Kidd |first10=Brian A. |last11=Gianettoni |first11=Jill |last12=Tersini |first12=Karen |last13=Utz |first13=Paul J. |last14=Valone |first14=Frank |last15=Steinman |first15=Lawrence |last16=Bht-3009 Study |first16=Group |title=Phase 2 trial of a DNA vaccine encoding myelin basic protein for multiple sclerosis |volume=63 |issue=5 |pages=611–20 |doi=10.1002/ana.21370 |journal=Annals of Neurology|display-authors=8 |citeseerx=10.1.1.418.3083 |s2cid=16111674 }}
• Profiling of antibody signatures{{cite journal |doi=10.1128/CVI.00208-09 |title=Peptide Microarray-Based Identification of Mycobacterium tuberculosis Epitope Binding to HLA-DRB1*0101, DRB1*1501, and DRB1*0401 |year=2009 |last1=Gaseitsiwe |first1=S. |last2=Valentini |first2=D. |last3=Mahdavifar |first3=S. |last4=Reilly |first4=M. |last5=Ehrnst |first5=A. |last6=Maeurer |first6=M. |journal=Clinical and Vaccine Immunology |volume=17 |pages=168–75 |pmid=19864486 |issue=1 |pmc=2812096}}{{Cite journal|last1=Weber|first1=Laura K.|last2=Palermo|first2=Andrea|last3=Kügler|first3=Jonas|last4=Armant|first4=Olivier|last5=Isse|first5=Awale|last6=Rentschler|first6=Simone|last7=Jaenisch|first7=Thomas|last8=Hubbuch|first8=Jürgen|last9=Dübel|first9=Stefan|date=April 2017|title=Single amino acid fingerprinting of the human antibody repertoire with high density peptide arrays|journal=Journal of Immunological Methods|volume=443|pages=45–54|doi=10.1016/j.jim.2017.01.012|issn=1872-7905|pmid=28167275}} and epitope mapping
• Finding neutralizing antibodies{{cite journal |pmid=21849452 |year=2011 |last1=Tomaras |first1=GD |last2=Binley |first2=JM |last3=Gray |first3=ES |last4=Crooks |first4=ET |last5=Osawa |first5=K |last6=Moore |first6=PL |last7=Tumba |first7=N |last8=Tong |first8=T |last9=Shen |first9=X |last10=Yates |first10=N. L. |last11=Decker |first11=J. |last12=Wibmer |first12=C. K. |last13=Gao |first13=F. |last14=Alam |first14=S. M. |last15=Easterbrook |first15=P. |last16=Abdool Karim |first16=S. |last17=Kamanga |first17=G. |last18=Crump |first18=J. A. |last19=Cohen |first19=M. |last20=Shaw |first20=G. M. |last21=Mascola |first21=J. R. |last22=Haynes |first22=B. F. |last23=Montefiori |first23=D. C. |last24=Morris |first24=L. |title=Polyclonal B cell responses to conserved neutralization epitopes in a subset of HIV-1-infected individuals |volume=85 |issue=21 |pages=11502–19 |doi=10.1128/JVI.05363-11 |pmc=3194956 |journal=Journal of Virology|display-authors=8 }}

• Identification of substrates for orphan enzymes{{cite journal |pmid=22205724 |year=2011 |last1=Kindrachuk |first1=J |last2=Arsenault |first2=R |last3=Kusalik |first3=T |last4=Kindrachuk |first4=KN |last5=Trost |first5=B |last6=Napper |first6=S |last7=Jahrling |first7=PB |last8=Blaney |first8=JE |title=Systems kinomics demonstrates congo basin monkeypox virus infection selectively modulates host cell signaling responses as compared to West African monkeypox virus |doi=10.1074/mcp.M111.015701 |journal=Molecular & Cellular Proteomics |volume=11 |issue=6 |pages=M111.015701|doi-access=free |pmc=3433897 }}
• Optimization of known enzyme substrates{{cite journal |doi=10.1074/jbc.M202042200 |title=Molecular Basis for the Substrate Specificity of NIMA-related Kinase-6 (NEK6). EVIDENCE THAT NEK6 DOES NOT PHOSPHORYLATE THE HYDROPHOBIC MOTIF OF RIBOSOMAL S6 PROTEIN KINASE AND SERUM- AND GLUCOCORTICOID-INDUCED PROTEIN KINASE IN VIVO |year=2002 |last1=Lizcano |first1=J. M. |journal=Journal of Biological Chemistry |volume=277 |issue=31 |pages=27839–49 |pmid=12023960 |last2=Deak |first2=M |last3=Morrice |first3=N |last4=Kieloch |first4=A |last5=Hastie |first5=CJ |last6=Dong |first6=L |last7=Schutkowski |first7=M |last8=Reimer |first8=U |last9=Alessi |first9=DR |doi-access=free }}
• Elucidation of signal transduction pathways{{cite journal |doi=10.1523/JNEUROSCI.3056-07.2007 |title=NMDA Receptor Activation Dephosphorylates AMPA Receptor Glutamate Receptor 1 Subunits at Threonine 840 |year=2007 |last1=Delgado |first1=J. Y. |last2=Coba |first2=M. |last3=Anderson |first3=C. N. G. |last4=Thompson |first4=K. R. |last5=Gray |first5=E. E. |last6=Heusner |first6=C. L. |last7=Martin |first7=K. C. |last8=Grant |first8=S. G. N. |last9=O'Dell |first9=T. J. |journal=Journal of Neuroscience |volume=27 |issue=48 |pages=13210–21 |pmid=18045915 |pmc=2851143 }}
• Detection of contaminating enzyme activities
• Consensus sequence and key residues determination{{cite journal |pmid=21756916 |year=2011 |last1=Thiele |first1=A |last2=Krentzlin |first2=K |last3=Erdmann |first3=F |last4=Rauh |first4=D |last5=Hause |first5=G |last6=Zerweck |first6=J |last7=Kilka |first7=S |last8=Pösel |first8=S |last9=Fischer |first9=G |last10=Schutkowski |first10=Mike |last11=Weiwad |first11=Matthias |title=Parvulin 17 promotes microtubule assembly by its peptidyl-prolyl cis/trans isomerase activity |volume=411 |issue=4 |pages=896–909 |doi=10.1016/j.jmb.2011.06.040 |journal=Journal of Molecular Biology|display-authors=8 }}
• Identifying sites for protein-protein interactions within a complex{{cite journal |pmid=23071676 |year=2012 |last1=Parsons |first1=LS |last2=Wilkens |first2=S |journal=PLOS ONE |volume=7 |issue=10 |pages=e46960 |doi=10.1371/journal.pone.0046960 |title=Probing subunit-subunit interactions in the yeast vacuolar ATPase by peptide arrays |pmc=3470569|bibcode=2012PLoSO...746960P |doi-access=free }}

Data analysis and evaluation of results is the most important part of every microarray experiment.{{cite journal |pmid=21621003 |year=2012 |last1=Hecker |first1=M |last2=Lorenz |first2=P |last3=Steinbeck |first3=F |last4=Hong |first4=L |last5=Riemekasten |first5=G |last6=Li |first6=Y |last7=Zettl |first7=UK |last8=Thiesen |first8=HJ |title=Computational analysis of high-density peptide microarray data with application from systemic sclerosis to multiple sclerosis |volume=11 |issue=3 |pages=180–90 |doi=10.1016/j.autrev.2011.05.010 |journal=Autoimmunity Reviews}} After scanning the microarray slides, the scanner records a 20-bit, 16-bit or 8-bit numeric image in tagged image file format (*.tif). The .tif-image enables interpretation and quantification of each fluorescent spot on the scanned microarray slide. This quantitative data is the basis for performing statistical analysis on measured binding events or peptide modifications on the microarray slide. For evaluation and interpretation of detected signals an allocation of the peptide spot (visible in the image) and the corresponding peptide sequence has to be performed. The data for allocation is usually saved in the GenePix Array List (.gal) file and supplied together with the peptide microarray. The .gal-file (a tab-separated text file) can be opened using microarray quantification software-modules or processed with a text editor (e.g. notepad) or Microsoft Excel. This "gal" file is most often provided by the microarray manufacturer and is generated by input txt files and tracking software built into the robots that do the microarray manufacturing.

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Category:Biological techniques and tools

Category:Immunology

Category:Microarrays

Category:Protein methods